title: Metabarcoding for NoV surveillance: Test Set Sample sequencing
description: Norovirus (NoV) specific metabarcoding was performed employing Illumina short-read sequencing. This amplicon-based approach takes advantage of the well-established primers used for NoV genotyping, that target specific, well-studied regions of the viral genome, namely portions of the genes encoding the RNA-dependent RNA polymerase (RdRp) and the capsid protein (VP1) (Imamura et al., 2017, Lun et al., 2018, Oshiki et al., 2018). In addition, primers that target a longer fragment (400-500 nt) comprising a part of RdRp and a part of VP1 gene sequences were also employed, thus allowing the typing of both genes in the same amplicon (dual typing). The PCR amplicons were tagged and indexed by an additional PCR, purified, quantified and mixed into a pool to perform sequencing on the Illumina Miseq platform. The raw reads, are thus, amplified genome fragment products that arise from different (Noro)viruses present in the sample. Raw reads were demultiplexed directly by the Illumina MiSeq sequencer. Reads quality was verified using FastQC (V.0.11.8 https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Andrew, 2010) and MultiQC (V.1.9 https://multiqc.info/ Ewels et al., 2010) tools. Reads were assembled into clusters using the FROGS suite in a Galaxy environment (Escudié et al, 2018). The sequence of each cluster was genotyped using the Norovirus Typing Tool 2.0 to allow for the characterization of the diversity of the NoV genotypes. Samples come from two different sequencing sets - Baseline Survey (BLS) samples, and two synthetic Test Set samples (TSS1 and TSS2). The TSS2 samples were prepared by oysters' bio-accumulation with selected human stool samples positive for NoV. The digestive tissues of the oysters were selected because it is known to concentrate most of the NoV contamination and is used in the standardized method for NoV and hepatitis A virus detection in shellfish (ISO 15216-1 2017). Digestive tissues from the laboratory-contaminated oysters were serially diluted in digestive tissues from oysters confirmed to be non-contaminated by norovirus, to obtain oyster samples with four levels of contamination: high (TSS2.1), intermediate (TSS2.2), medium-low (TSS2.3) and very-low (TSS2.4). In addition, TSS2 contained a negative control consisting in digestive tissue from NoV-negative oyster used for dilution (TSS2.5) and a background control consisting in digestive tissue from oysters used for TSS2 preparation, before bio-accumulation (TSS2.0). For the preparation of TSS2, in addition to the selection criterion for TSS1, the quantity of human stool sample available was a limiting factor. Indeed, for artificial contamination of oysters, the in-house protocol used around 10^7 NoV genome copies per strain, which ensures that oysters are efficiently and homogeneously contaminated, taking into account the loss of virus binding on oyster shells and tank walls (Maalouf et al., 2011). Thus, TSS2 contains 6 NoV strains, 4 strains belonging to GII (GII.17[P17], GII.3[P21], GII.4[P31], GII.2[P16]) and 2 belonging to GI (GI.4[P4], GI.1[P1]). Strains belonging to NoV GII.4[P16], GII.4[P31] and GII.17[P17] genotypes were selected in priority due to their high frequency in the European human population since 2016 (Noronet unpublished data, search date 19-01-2019) (https://www.erasmusmc.nl/en/research/projects/Noronet).